Method for recovering sperm nucleic acid from a forensic sample

ABSTRACT

A method for selectively recovering nucleic acid from a sperm cell in a sample containing cells of at least a sperm cell and an epithelial cell, and a cell suspension medium comprising extracellular impurities, is provided. The method entails introducing a sample into a vessel, sequestering the cells from the remaining sample components, washing the cells with a washing solution either before or after sequestration, removing the impurities-containing cell suspension medium from the vessel while retaining the cells; lysing selectively cells of the first cell type; and isolating the nucleic acid from the lysed cells. Methods for recovering nucleic acid from the second cell type are also provided.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/961,734, filed Jul. 23, 2007.

FIELD OF THE INVENTION

The invention relates to the field of isolating cells from a samplecontaining cells and non-cell impurities and further isolating nucleicacid from a specific cell type.

More specifically, the invention relates to the field of isolating spermcells from a sample containing extracellular impurities and at least oneother type of contaminating cell (e.g., epithelial cell) and recoveringDNA from the sperm cells and nonsperm cells for various purposes,including forensic purposes.

BACKGROUND OF THE INVENTION

Forensic DNA analysis of sexual assault evidence often involves analysisof DNA from sperm cells and DNA from other cells such as epithelialcells. Sperm cells are normally obtained from a rape victim by rubbing aswab against a mucous membrane. The samples obtained from victims oftencontain a mixture of sperm and epithelial cells. Because the epithelialcells may outnumber sperm cells in the sample by at least an order ofmagnitude, the former can cause contamination of sperm cell DNA, whensperm DNA is purified. Consequently, it is often desirable to separate,as cleanly as possible, the sperm cells and epithelial cells, or thesperm DNA and the epithelial DNA, prior to analysis. Separation andisolation of DNA from sperm and epithelial cells are essential steps inidentifying an assailant from a forensic specimen, and in associatingthe assailant with the victim.

The standard method for purifying sperm from swabs is based ondifferential extraction. Separation of the sperm DNA from the victim'sDNA removes ambiguity, facilitates DNA analysis and allows for easierinterpretation of the assailant's DNA profile in a rape case. Althoughdifferential extraction is commonly used to separate sperm andepithelial cells, the standard protocol is time consuming and laborious,and entails epithelial cell lysis prior to sperm cell lysis.

Typically, cells are first resuspended from a forensic specimen,followed by selective digestion of the victim's epithelial cells with asolution containing Proteinase K and SDS (sodium dodecyl sulfate). Theintact sperm are separated from the solubilized, contaminating DNA andepithelial cell debris by centrifugation, careful removal ofsupernatant, and extensive washing of the sperm pellet (see e.g., Giustiet al., J. Forensic Sci., 31:409-417, 1986; Gill et al. Nature318:577-579, 1985; Wiegand et al., Int J. Legal Med., 104:359-360, 1992;and Yoshida et al., Forensic Sci. Int., 72:25-33, 1995). Unfortunately,the processes of centrifugation and careful removal of supernatant aredifficult to automate and can cause the loss of sperm DNA due tomultiple sample handling steps.

In one example of this procedure, Gill et al. (supra) describe a processfor isolating sperm DNA from vaginal swabs taken from sexual assaultvictims. These swabs contain sperm and also a large excess of thevictim's epithelial cells. The epithelial cells and the DNA contained inthese cells are removed by preferential lysis (i.e., by incubation ofthe cell mixture in a buffer solution containing SDS, and Proteinase K).Sperm nuclei are impervious to this treatment because they havedisulfide bond cross-linked thiol-rich proteins, while other cell typesare digested and the corresponding DNA is solubilized. Afterpreferential lysis, the samples are centrifuged to separate the spermnuclei from the victim's solubilized DNA. The supernatant containing thevictim's DNA is removed and the sperm pellet is washed repeatedly. Thesperm nuclei are subsequently lysed by treatment with a buffer solutioncontaining SDS, proteinase K and DTT (dithiothreitol), and the lysateseparated from the contaminating cells by centrifugation.

Wiegand et al. (supra) attempted to improve on the method of Gill et al.for samples having low sperm counts by using mild lysis conditions andby avoiding the washing steps.

A number of proposals for separating sperm cells from epithelial cellsare based on filtration. Chen et al. (J Forensic Science 43:114-118,1998) and Garvin (PCT/US01/01835) separate the sperm from the epithelialcells before differential lysis by gravitational or mild vacuumfiltration or by use of a filter material that can withstand strongvacuum or centrifugal forces without having the pores increase in size.DNA is then isolated from the sperm collected in the filtrate.

Differential extraction, specifically for sperm cell analysis, has beencarried out by first lysing epithelial cells, before extracting the DNAfrom sperm. In order to reduce the time for forensic analysis, it isdesirable to selectively extract the DNA from the sperm cells,initially. In addition, because the selective epithelial cell lysisconditions also cause some sperm lysis, lysing epithelial cells beforethe sperm cell fraction reduces the number of sperm cells present in thesample. The additional wash steps that are required to remove epithelialDNA also contribute to the loss of sperm cells.

Accurate sperm analysis can be impeded by the lysing or presence ofepithelial cells, or other contaminating cell types, such as leukocytes.DNA present in the forensic sample, from damaged nonsperm cells, cancontaminate the cells (most likely by binding to the surface of spermcells), which can inhibit accurate identification of a perpetrator,especially when the number of recovered sperm cells is low.

The present inventor discovered that although techniques exist fordifferential extraction of one type of nucleic acid (from an individualcell type) from a heterogeneous cell population, residual contaminatingnucleic acid is still a problem that warrants more precise extractionmethodologies. One application where the need exists for highly specificdifferential extraction is forensic, specifically sexual assaultanalysis.

Because of their fragile nature, epithelial and other cells, e.g.,leukocytes, in a sexual assault sample often break and lyse duringhandling, sample processing or storage. This residual DNA, if notremoved, may contaminate male DNA during selective sperm lysis,especially when the number of sperm cells is low. In addition,epithelial and sperm cell surfaces are highly functionalized, withmolecules (e.g., glycoproteins) that bind nucleic acid through ionicinteractions, as well as other non-covalent methods. These cell surfaceproteins have been found to associate with extracellular nucleic acidpresent in a sample.

SUMMARY OF THE INVENTION

The present invention serves to reduce the amount of residual DNA andcontaminating cells, e.g., leukocytes, present in a forensic sample, byimplementation of a novel wash step, prior to sperm cell lysis.

The present invention can be employed with classical centrifugationbased methods, size exclusion filtration, in addition to particle- orbead-based methodologies for isolating cells from a sample. Themethodology is readily adaptable to automation and high throughput withexisting equipment such as the Beckman Biomek® (Beckman Coulter,Fullerton, Calif.) or a Tecan liquid automated handling system (e.g.,Freedom Evo® Services Tecan Systems, San Jose, Calif.). The invention ismore widely applicable to both forensic and other situations wherenucleic acid has to be isolated from one type of cell found in a mixtureof more than one type of cell, as long as each cell type can beselectively lysed (except the last remaining cell type which need not beselectively lysed).

The present invention, in one aspect, concerns a method for selectivelyrecovering DNA from sperm cells in a sample containing mixed sperm cellsand epithelial cells and a cell suspension medium comprising impurities.The impurities include contaminating residual DNA and othercontaminating cells that may be present, such as leukocytes. In thisaspect, the method entails isolating the mixed cells from the medium, ina first isolating step, lysing selectively the sperm cells, andrecovering the DNA from the sperm cells. The method also includes atleast one of two additional steps, wherein the first step is a samplewash with a first washing solution that disengages or digests residualDNA from the surface of the cells prior to the first isolating step. Thesecond step includes a wash/isolating procedure that comprises washingthe isolated mixed cells with a second washing solution followed by theisolation of the washed mixed cells from the second washing solution ina second isolating step, prior to the lysing step. The first or secondwashing solution selectively lyses leukocytes, if leukocytes are presentin the sample. It should be noted that since the second isolating stepmight not be performed (in the event there is no wash step after thefirst isolating step) there could be only one isolating step in theprocess.

The present inventor found that this simple wash or washes effectivelyreduces the problem of residual DNA contamination such that it will notinterfere with genotyping without resorting to expensive, or timeconsuming or manipulative steps that could introduce other problems.

In a further aspect, the cells are isolated by either size exclusionfiltration, by centrifugation, or with magnetic particles.

In another aspect, a method is provided for selectively recovering DNAfrom sperm cells in a sample containing mixed sperm cells and epithelialcells and a cell suspension medium comprising impurities, wherein theimpurities include contaminating extracellular material such as residualDNA and possibly other contaminating cells such as leukocytes. Themethod entails introducing the sample onto a size exclusion filter,including any first washing solution, isolating the mixed cells from thecell suspension medium, in a first isolating step, by sequestering thecells on the filter and allowing the cell suspension medium to form afiltrate, lysing selectively the sperm cells, and recovering the DNAfrom the sperm cells. The method also includes at least one of twoadditional steps, wherein the first step is a sample wash that includeswashing the sample by mixing it with a first washing solution to form awashed sample, prior to introducing the sample onto the size exclusionfilter. The second step includes a wash/isolating procedure thatcomprises washing the isolated mixed cells with a second washingsolution followed by the isolation of the washed mixed cells from thesecond washing solution in a second isolating step, prior to the lysingstep. The first or second washing solution serves as a selectiveleukocyte lysis buffer, if leukocytes are present in the sample.

In yet another aspect of the invention, a method for selectivelyrecovering nucleic acid from sperm cells in a sample containing mixedsperm cells and epithelial cells and a cell suspension medium comprisingimpurities is provided. The impurities include contaminating residualDNA and possibly other contaminating cells such as leukocytes. Themethod entails centrifuging the sample, including any first washingsolution, to form a cell pellet and a supernatant containing thesuspension medium, including any first washing solution, therebysequestering the mixed cells from the suspension, followed by isolatingthe mixed cells from the supernatant in a first isolating step, lysingselectively the sperm cells; and recovering DNA from the sperm cells.The method also includes at least one of two additional steps whereinthe first step is a sample wash step that comprises mixing the samplewith a first washing solution to form a washed sample. The second stepis a wash/isolation procedure and comprises washing the isolated mixedcells with a second washing solution followed by centrifuging the washedmixed cells and second washing solution to form a pellet and asupernatant, thereby sequestering the washed cells from the suspension,followed by isolating the mixed washed cells from the supernatant, in asecond isolating step, prior to said lysing step. The first or secondwashing solution can selectively lyse leukocytes, if leukocytes arepresent in the sample.

In one aspect, the first washing solution or the second washing solutionor both washing solutions independently comprise one or a combination ofcationic salt such as MgCl₂, NaCl, KCl; polycation such as poly-lysine,polyanion such as heparin, all of which can interfere with DNA-cellsurface interaction. Salt solution such as MgCl₂ can also lyseleukocytes by imposing osmotic pressure, or disrupting leukocytemembrane and chromatin structure. In various aspects, DNase, which candigest extraneous DNA, is employed as a wash solution or portionthereof.

In a particular aspect of the invention, the sample comprises a forensicspecimen, which can include a specimen taken from a sexual assaultvictim. The cell types in such a sample include at least sperm andepithelial cells and sometimes leukocytes as well. In principle, thenucleic acid in a sample can be DNA or mRNA or total RNA. Commonly, itis DNA.

In another aspect of the invention, the isolated sperm nucleic acid canbe used for downstream steps, including, but not limited to, quantifyingthe purified nucleic acid, amplifying the nucleic acid and separatingthe amplified short tandem repeat fragments for genotyping.

DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present invention in any way.

FIG. 1 is a process flow chart showing steps to isolate and recover DNAfrom sperm cells in a sample containing both sperm and epithelial cells,in accordance with some embodiments of the present invention.

FIGS. 2A-2D are STR typing profiles of sperm DNA isolated with a methodof the invention as illustrated in Example 1. Primers for the variousloci were labeled with a fluorescent dye, followed by amplification ofthe various loci and separation of products by electrophoresis.Fluorescence excitation/emission wavelengths and dyes employed were494/522 nm, 6-FAM (FIG. 2A), 538/554 nm, VIC (FIG. 2B), 546/575 nm, NED(FIG. 2C), 558/595 nm, PET (FIG. 2D).

FIG. 3 is a bar graph showing the effect of MgCl₂ concentration (washingsolution) on the amount of residual epithelial cell DNA present in asample.

FIGS. 4A-4D are STR typing profiles of sperm DNA isolated with a methodof the invention as illustrated in Example 3. Primers for the variousloci were labeled with a fluorescent dye, followed by amplification ofthe various loci and separation of products by electrophoresis.Fluorescence excitation/emission wavelengths and dyes employed were494/522 nm, 6-FAM (FIG. 4A), 538/554 nm, VIC (FIG. 4B), 546/575 nm, NED(FIG. 4C), 558/595 nm, PET (FIG. 4D).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION Definitions

The term “nucleic acid,” as used herein, encompasses DNA and RNAmolecules, as well as DNA/RNA chimeras and nucleic acid molecules withunnatural bases and/or sugar moieties. In a particular forensicembodiment, DNA from sperm cells of the assailant is isolated from DNAof nonsperm cells (usually epithelial cells) of the victim.

The “sample” used in the present invention can be any heterogeneouscellular suspension containing two or more cell types wherein at leastone of the types of cells can be selectively lysed. The sample alsoincludes suspension medium such as reconstituting buffer which alsocontains impurities from the sample, e.g., extraneous DNA. In someembodiments, the sample is reconstituted from a forensic specimen, andcan optionally include the swab or cotton applicator on which theforensic specimen is collected. It will be understood that after awashing solution is combined with the sample, the sample will alsocomprise a washing solution. Thus, separate recitation of the washingsolution is not necessary.

The term “forensic specimen,” as used herein, is a specimen obtained toaddress legal issues, including, but not limited to, murder, rape,trauma, assault, battery, theft, burglary, other criminal matters,identity testing, paternity testing, and mixed application samples. Itbroadly refers to a substrate which contains a specimen of biologicalmaterials such as blood, blood stains, saliva, skin debris, feces, fecesstains, urine, sperm cells, epithelial cells, muscle tissue, bone ormuscle remains, or mummified remains. In some embodiments, the “forensicspecimen” is contained on, and includes, a swab or cotton applicator onwhich the biological specimen is collected.

The term “differential extraction,” as used herein, refers to extractionmethods used to separate nucleic acid from individual cell types withina heterogeneous population of cells, such as the selective lysis ofsperm cells in an epithelial-sperm cell mixture.

The two or more “cell types” amenable for use in embodiments of thepresent invention can include any two or more of the following, as longas at least one cell type in the mixture can be selectively lysed: spermcells, epithelial cells, erythrocytes, platelets, neutrophils,lymphocytes, monocytes, eosinophils, basophils, adipocytes,chrondrocytes, tumor cells, neurons, glial cells, astrocytes, red bloodcells, leukocytes, macrophages, hair cells, bladder cells, kidney cells,retinal cells, rod cells, cone cells, antigen presenting cells, T-cells,B-cells, plasma cells, muscle cells, ovarian cells, prostate cells,vaginal epithelial cells, testicular cells, sertoli cells, lutein cells,cervical cells, endometrial cells, mammary cells, follicle cells, mucouscells, ciliated cells, nonkeratinized epithelial cells, keratinizedepithelial cells, lung cells, goblet cells, columnar epithelial cells,squamous epithelial cells, osteocytes, osteoblasts and osteoclasts.

In certain embodiments, more than two cell types are present in thesample. In certain forensic embodiments, sperm cells and epithelialcells are essentially the only cell types present in the sample. Incertain forensic embodiments, sperm cells, epithelial cells andleukocytes are the cell types present in the sample. In embodimentswhere leukocytes are present, the first wash step acts to lyse theleukocytes selectively, while keeping the sperm and epithelial cellsintact.

“Sperm cell,” as used herein, can include an intact sperm cell oressentially intact sperm cell, (e.g., a sperm cell that has lost itsflagellum or tail) as long as the nucleus is still intact.

The term “cell mixture,” as used herein, refers to a heterogeneouscollection of at least two different cell types.

The “cell suspension medium” is a buffer, or liquid, in which the cellmixture is present. The cell suspension medium can be a reconstitutingbuffer, if the cell mixture was originally present on a solid substrate,or a washing solution, if isolated cells are resuspended in it. Thereconstituting buffer can be, by way of nonlimiting example, 1×Phosphate-Buffered Saline (PBS). Cell suspension media are well-knownand can be readily selected by those skilled in the art. For thepurposes of this invention, a solution added to a cell suspensionmedium, e.g., washing solution, is part of the cell suspension medium,even if it is not originally present.

The term “supernatant,” as used herein, describes the liquid, or buffer,present in the vessel, after cells and particles have settled to thebottom, or side, of the vessel. The supernatant can comprise impurities,such as DNA from compromised cells, or fibers from a swab, if the cellmixture was originally reconstituted from a solid substrate, or celllysate after a lysis step has occurred, or spent washing solution aftera wash step has occurred. Additionally, once the cells and particleshave settled to the side or bottom of a vessel, the cell suspensionmedium can be considered a supernatant, and can comprise impurities.Thus, the usage and meaning of cell suspension medium and supernatantoverlap.

“Residual DNA,” as used herein, refers to extracellular DNA in thesample medium and not present within a cell, e.g., in a sperm-epithelialcell mixture, DNA from compromised or lysed cells. Residual DNA may bebound to or otherwise become associated with a cell's surface andpotentially contaminate DNA extracted from the cell. In embodimentswhere leukocytes are present, residual leukocyte DNA is formed duringthe first wash step because the first wash step lyses the leukocytes.

A “vessel,” for use with the present invention, can be any tube (e.g.,0.2 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL in volume), container, or well, whichis open on the top, and enclosed on all sides and the bottom. Includedin this definition are reagent cartridges, designed for automatedequipment. Multiple wells can be joined together to form a plate, suchas a 96-well plate or any other plate adapted to be used in automatedequipment.

The term “particles responsive to a magnetic field,” as used herein, isintended to encompass all particles (e.g., beads or irregular shapeparticles) that can capture, i.e., interact or associate with, cells ornucleic acid or both (under different buffer conditions) to accomplishsequestration of the cells from the medium, and which migrate whenplaced within a magnetic field. In some embodiments, more than oneparticle can associate with one another to form particle aggregates. Inother embodiments, the particles are uniform in diameter and/or shape,e.g., beads. When a magnet or magnetic field is in close proximity to,or in contact with, the vessel, the particles in the reaction vesselmigrate towards the source of the magnetic field. Included areferromagnetic, paramagnetic, and superparamagnetic particles.Nonlimiting examples of commercially available particles responsive to amagnetic field are particles comprised of porous silica withsupermagnetic core MP-50 (6.5 μm), MP-85 (>8 μm) (W.R. Grace, Columbia,Md.), iron oxide immobilized with streptavidin (Sigma, St. Louis, Mo.),and iron(III) oxide powder (5 μm) (Sigma, St. Louis, Mo.), DNA IQ™silica particles (Promega, Madison, Wis.), MagPrep® silica particles(Novagen, San Diego, Calif.), BcMag® silica-modified magnetic beads (5μm or 1 μm) (Bioclone Inc., San Diego, Calif.), supermagnetic silicaparticles (1 μm or 0.75 μm, G. Kisker GbR, Steinfurt, Germany), andDynabeads®(Invitrogen, Carlsbad, Calif.) with different type of surfacefunctional groups (e.g., Dynabeads® MyOne carboxylic acid beads,Dynabeads® WCX, Dynabeads® TALON and Dynabeads® MyOne tosylactivated).The terms “particles,” or “magnetic particles,” as used herein, have thesame meaning as “particles responsive to a magnetic field” and aresimply shorthand for the longer term.

“Size exclusion filtration,” as used herein, refers to a centrifugationprocess wherein a vessel containing a filter is subjected tocentrifugation. The sample, lysis buffer, or washing solution is appliedto the filter, optionally mixed with cells sequestered on the filter,and centrifuged. The filter allows components smaller than the size ofthe filter pore to be isolated (in the “filtrate”) from componentsbigger than the size of the filter pore (i.e., cells). One example of asuitable spin filter is the Corning spinX Costar 8160 filter, with apore size of 220 nm, available from Sigma (part # CLS8160). Other spinfilters with similar pore sizes can also be employed. The pore size ofthe filter should not be larger than 2 μm, in order to keep epithelialcells from passing into the filtrate.

The term “short tandem repeat,” or “STR,” as used herein, refers to allsequences between 2 and 7 nucleotides long which are tandemly reiteratedwithin a segment of the human genome.

DNA typing (or “genotyping”) involves the analysis of alleles of genomicDNA with characteristics of interest, commonly referred to as “markers.”Most typing methods in use today are specifically designed to detect andanalyze differences in the length and/or sequence of one or more regionsof DNA markers known to appear in at least two different forms in apopulation. Such length and/or sequence variation is referred to as“polymorphism.” Any region (i.e., “locus”) of DNA in which such avariation occurs is referred to as a “polymorphic locus.”

Genetic markers which are sufficiently polymorphic with respect tolength or sequence have long been sought for use in identityapplications, such as paternity testing and identification of tissuesamples collected for forensic analysis. The discovery and developmentof such markers and methods for analyzing such markers have gone throughseveral phases of development over the last several years.

In recent years, the discovery and development of polymorphic shorttandem repeats (STRs) as genetic markers has played an important role inDNA typing. In this approach, amplified alleles at each selected locusmay be differentiated based on length variation. Amplification protocolswith STR loci can be designed to produce small products, generally from60 to 500 base pairs (bp) in length, and alleles from each locus areoften contained within a range of less than 100 bp. This allowssimultaneous electrophoretic analysis of several systems on the same gelor capillary electrophoresis by careful design of PCR primers such thatall potential amplification products from an individual system do notoverlap the range of alleles of other systems. These results can then beused for example to identify the parentage of human children, and toidentify the source of blood, saliva, semen, and other tissue found at acrime scene or other sites requiring identification of human remains.

The term “selective sperm lysis buffer,” as used herein, refers to abuffer that is capable of preferentially lysing sperm cells in a mixturecomprising sperm cells and at least one other type of non-sperm cells(that are not susceptible to lysis by this buffer). “Preferentiallylysing sperm cells” as used herein, refers to the lysis of sperm cells,whereas nonsperm cells are not lysed. Quantitatively, at least 80%, 85%,90%, 95%, or 99% of the sperm cells are lysed, whereas at least 80%,85%, 90%, 95% of the nonsperm cells are not lysed. In certainembodiments, only a negligible number of non-sperm cells are lysedtogether with the sperm cells. Nonlimiting examples of such buffers area combination of 880 mM NaCl and 85 mM DTT, and are further describedinfra, specifically in Example 1. Selective sperm lysis buffers aredisclosed in U.S. Prov. App. No. 60/899,106, incorporated herein byreference in its entirety. In some embodiments, selective sperm celllysis buffers comprise at least one disulfide bond reducing reagent(e.g., dithiothreitol (DTT), tris(2-carboxyethyl)phosphine HCl (TCEP),mercaptoethanol (ME), glutathione (GSH)) and at least one salt reagent(e.g., sodium chloride (NaCl), potassium chloride (KCl), lithiumchloride (LiCl), magnesium chloride (MgCl₂), magnesium sulfate (MgSO₄),sodium nitrate (NaNO₃), calcium chloride (CaCl₂), calcium sulfate(CaSO₄)). In certain embodiments, a selective sperm lysis buffercomprises at least one disulfide bond reducing reagent with aconcentration of at least 0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M,0.7M or 0.8M. In certain embodiments, a selective sperm lysis buffercomprises at least one salt reagent with a concentration of at least0.1M, 0.25M, 0.5M, 1M, 1.5M, 2M or higher.

The term “washing solution,” as used herein, refers to a reagent with afunction of removing residual DNA and other impurities contained in thesupernatant from cells. In addition, the “washing solution” acts as aselective leukocyte lysis buffer, when leukocytes are present in asample with sperm and epithelial cells. Washing solution can be, but isnot limited to, water, 1×PBS, salt solutions such as MgCl₂, NaCl and KCldiluted in an appropriate buffer, e.g., water, 1×PBS, Tris-HCl, or otherphysiologically acceptable solution. In some embodiments, the washingsolution is MgCl₂ diluted in 1×PBS.

In some embodiments, the washing solution is or comprises a DNase in itsreaction buffer. In some embodiments, DNase is a component of thewashing solution. In embodiments where DNase is a component of thewashing solution, DNase can be added in conjunction with a cationicsalt, such as MgCl₂. In yet other embodiments, a wash with a cationicsalt can be immediately followed with a DNase wash (see, e.g., example 3below). The two washes carried out in Example 3 correspond to theemployment of steps 2 and 4 in FIG. 1.

In some embodiments, the forensic specimen is reconstituted directly ina washing solution.

DNase is salt sensitive and optimal DNase activity occurs in low saltsolution (<20 mM). When wash solution containing high saltconcentration, additional amount of DNase can be used to compensate forthe reduced enzyme efficiency. A serial wash with a salt washing bufferfollowed by DNase should not hinder the enzyme's activity.

In some embodiments, the first or second or both washing solutionscomprise DNase. In particular forensic embodiments, 0.01 U to 10 U ofDNase is present in the washing solution. In embodiments where DNase isin the presence of a salt solution, up to 50 U of DNase can be employed.

In some embodiments, a washing solution can comprise a polyanion, suchas heparin polyanion. Other examples of polyanions include poly(acrylicacid), poly(methacrylic acid), poly(styrene sulfonate),poly(2-acrylamido-2-methyl-1-propane-sulfonic acid) (PAMPS), or NAFION®(DuPont™). In some embodiments, the washing solution can comprise apolycation, such as poly-lysine, or a combination of polycations. Still,in some embodiments, the wash solution can comprise DNA from anon-primate source, such as salmon sperm DNA, to compete the female DNAfrom a sperm cell's surface. The washing solution can be combined withthe sample medium or the medium can be removed and the washing solutionfreshly added. In some embodiments, forensic specimen is incubated inthe wash solution for up to 24 hours before separating the mixed spermand epithelial cells from the supernatant (which may contain leukocytelysate).

When polyanion or polycation are included in the washing solution, themolar concentration of the polyanion or polycation should be in largeexcess to that of extraneous DNA molecules, in order to ensure that DNAbound to a cell's surface is replaced by polyanion or polycation. Atypical forensic sample contains about 10 ng of extraneous DNA (Anal.Chem. 2005, V. 77, pp. 742-749.). Assuming final volume of wash solution(with sample) is 500 μL, and DNA has been fragmented with a averagelength of 500 bp (the largest STR amplicon is about 500 bp), the molarconcentration of DNA is about 60 pM. Based on estimated DNAconcentration, one can select and optimize the optimal polyanion orpolycation concentration for a particular washing solution. In addition,once the amount of extraneous DNA in the sample is known, one can alsodetermine the amount of DNase needed for complete digestion ofextraneous DNA for a given digestion time and temperature (e.g., roomtemperature, 37° C.). For example, one unit of Turbo DNase is defined asthe amount of Turbo DNase required to completely degrade 1 μg of DNA in10 min at 37° C. Therefore, one unit of Turbo DNase should be able tocompletely degrade all the extraneous DNA found in a typical sexualassault sample. However, excess amount of Turbo DNase (for example, 5units or more) could be used to achieve shorter DNA digestion timeand/or room temperature operation. When another type of DNase is usedfor the purpose of digesting extraneous DNA in a sexual assault sample,the optimal digestion condition (amount of DNase, digestion time andtemperature) can be easily optimized experimentally.

“Nucleic acid capture particles” can be any particles that areresponsive to a magnetic field which also bind to nucleic acid presentin the sample or supernatant, under specific conditions (e.g., anappropriate buffer for nucleic acid capture). The “nucleic acid captureparticles” can bind DNA or RNA preferentially, or all nucleic acid. Forexample, nucleic acid binds to silica magnetic particles in chaotropicbuffer (U.S. Pat. No. 5,234,809, incorporated in its entirety byreference). Buffers that can be employed for nucleic acid capture, withnucleic acid capture particles, can be solutions prepared from guanidiumisothiocyanate (GuSCN) and guanidium chloride. Nucleic acid can also becaptured by magnetic particles in solution containing high salt andalcohol (see, e.g., U.S. Pat. Nos. 5,523,231 and 5,705,628, bothincorporated herein, in their entirety, by reference). In someembodiments sodium acetate salt (2.5 M, pH 5,2) is used in conjunctionwith 70% ethanol. Other nucleic acid capture particles use bufferscomprising 0.5 M to about 5.0 M salt combined with 7%-13% polyethyleneglycol can be harnessed as a reaction buffer with capture particles.Salts such as sodium chloride, lithium chloride, barium chloride,potassium chloride, calcium chloride, magnesium chloride and cesiumchloride can all be used.

The term “elution buffer,” or “elution solution,” as used herein, refersto a reagent with a function of disrupting or breaking the interactionof nucleic acid with nucleic acid capture particles. For example, thebuffer can be any low salt solution (e.g., tris (10 mM)-EDTA (0.1 mM)(TE) buffer) or DNase-free water. Elution buffers are commonly employed,and are known in the art. Other low salt buffers (neutral to slightlybasic pH) can also be used.

SPECIFIC EMBODIMENTS

By way of overview and introduction, the process of the presentinvention is given as FIG. 1. A sample comprising a mixed cellsuspension, for example a forensic specimen, is provided at step 1. Thesample can then washed with a washing solution at step 2. If leukocytesare present in the sample, they are lysed during the first wash step(either step 2 or 4), while keeping the sperm and epithelial cellsintact. The cells in the sample are then isolated by a method chosen bythe user at step 3, for example by size exclusion filtration (3.1),magnetic particles (3.2), centrifugation (3.3), or any other methodknown in the art. The isolated mixed cells can then washed with awashing solution at step 4. If the wash at step 2 is implemented, thewash at step 4 is not required. Similarly, if the wash is employed atstep 4, the wash at step 2 is not required. However, both can beperformed if desired (see Example 3). At step 5, sperm cells areselectively lysed, for example with the sperm lysis buffer disclosed inU.S. Provisional App. Ser. No. 60/899,106, the entire contents of whichare incorporated by reference. Selective sperm lysis is followed byrecovery of the sperm cell lysate, which contains sperm DNA, at step 6.Sperm DNA can then be optionally further isolated from the sperm celllysate at step 7. These steps are discussed in turn.

A sample, if provided for example as a forensic specimen on a swab, isreconstituted prior to step 2, to form a cell suspension, for example,in a buffer comprising 1×PBS. In a particular forensic embodiment, aforensic specimen is reconstituted in the same vessel used for DNAisolation. In another forensic embodiment, a forensic specimen isreconstituted in a different vessel, and the cell suspension istransferred to the DNA isolation vessel. The vessel can contain magneticparticles or a size exclusion filter, or neither.

The swab itself can be removed, pushed aside, or it (or swab debris leftafter removal of the swab) can be digested using cellulose digestingenzymes. Examples of cellulose digesting enzymes include cellulase,beta-glucanase and those isolated from fungal sources such asAspergillus niger, Trichoderma reesei, and Trichoderma viride. Althoughenzymes of this type can affect epitope stability on cell surfaceproteins, the cell membranes, and DNA content of the cells, willgenerally not be compromised. Nevertheless, such enzymes are alsoconsidered impurities and should be removed. The first washing step ofthe present process can help accomplish this.

In various embodiments, a forensic sample is reconstituted in about 1 mLreconstituting buffer (e.g., 1×PBS) and gently mixed in a vessel (whichmay or may not be the vessel used for cell sequestration anddifferential extraction). The substrate (e.g., a swab) is then removedfrom the vessel and the cells from the specimen centrifuged. Thereconstituting buffer is then drawn off the cell pellet, and the cellpellet resuspended in 50 μL reconstituting buffer. In anotherembodiment, about 50 μL of the cell suspension medium is left on thecell pellet, with the rest of the medium drawn off, and the cell pelletreconstituted in the original medium (i.e., the 50 μL).

In some embodiments, including various forensic embodiments, a portionof the cell suspension can be used to count the number of cellsoriginally present in the sample. The number of cells present can givethe user of the method a basis for determining the quantity and/orconcentration of particular reaction component to employ.

Once the cell sample is present in solution, it can be washed bycombining the cell suspension with a washing solution (step 2). Inpreferred embodiments, the ratio of wash buffer to cell suspension is10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1 or 3:1. Optionally, after the cellsuspension is formed (either by initially providing mixed cells insolution, or by reconstituting a sample), it can be centrifuged andresuspended in resuspension buffer (e.g., 1×PBS), to form a moreconcentrated suspension. In preferred embodiments, the cell suspensionused throughout the process of the invention has a volume ranging from50 μL to 2 mL.

At step 2, the sample can be washed by combining a wash solution withthe cell suspension, followed by mixing the two components. Mixing canbe accomplished by pipetting, vortexing or shaking the vessel, e.g.,with an orbital shaker or by manual rotation. The two components canalso be mixed by passive diffusion. The cell suspension wash can becarried out in a vessel separate from the one used for sperm cell lysisand DNA isolation, or in the same vessel. Washing solutions aredescribed supra. Samples in the wash solution can be incubated for up to24 hours, if desired.

In some embodiments, a forensic specimen on a swab, applicator, orportion thereof, is reconstituted directly in a wash solution. Incertain forensic embodiments, the swab itself is removed afterincubation in the wash solution. In certain forensic embodiments, theswab is not removed after incubation in the wash solution. In aparticular forensic embodiment, a forensic specimen is reconstituted inthe same vessel used for DNA isolation. In another forensic embodiment,a forensic specimen is reconstituted in a different vessel, and the cellsuspension is transferred to the DNA isolation vessel. The DNA isolationvessel can contain magnetic particles or a size exclusion filter, orneither.

In some embodiments, including various forensic embodiments, a portionof the cell suspension can be used to count the number of cellsoriginally present in the sample. The number of cells present can givethe user of the method a basis for determining the quantity and/orconcentration of particular reaction component to employ.

Various cell isolation procedures can be employed at step 3, dependingon the preference of the user of the method. For example, the cells inthe cell suspension can be isolated by particles responsive to amagnetic field (3.2), as fully described in U.S. Provisional App. Ser.No. 60/890,460, a copy of which is attached as Appendix A and which isincorporated by reference herein in its entirety. Briefly, while thevessel, containing the cell suspension is in the presence of a magneticfield, the particles push the cells towards the source of the magneticfield and the cell suspension medium along with the impurities isremoved, whereas the particles and cells remain in the vessel. Theremoval of the cell suspension medium can be accomplished in anautomated fashion or manually, by pipetting or aspirating, both gentletechniques that avoid damage to the cells.

Although ionic interaction and/or hydrophobic interaction contribute tothe cell/particle(s) association, cell and particle association can alsobe established by physical trapping or temporary attachment of particlesto the cell under magnetic force if particles are smaller than cells, orphysical trapping or temporary attachment of cell to particle orparticle aggregates under magnetic force if cells are smaller than theparticle or particle aggregates. Other particle embodiments can employparticles that sequester cells based on other non-covalent interactions.Some non-covalent interactions that can be employed are hydrogenbonding, cation-π, π-π interactions, dipole-dipole,dipole-induced-dipole, charge-dipole and van Der Waals interactions.

The particles that will sequester sperm and epithelial cells willtypically have a diameter within the range of 0.5-100 μm; preferably1-10 μm with sizes outside the broader range not being excluded. Theexact limits of the size range can be ascertained for a given situationof particles and cells by routine experimentation.

In various embodiments, the cells are initially sequestered at step 3.3from the impurity containing cell suspension medium by centrifugation(to form a cell pellet). The cell suspension medium (i.e., thesupernatant) is then removed either in an automated fashion, or manually(e.g., by pipetting), to isolate the cells from the sample (containingresidual DNA from damaged epithelial cells). Instruments amenable foruse with the present invention include, but are not limited the BeckmanBiomek®(g (Beckman Coulter, Fullerton, Calif.) or a Tecan liquidautomated handling system (e.g., Freedom Evo® Services Tecan Systems,San Jose, Calif.).

In other embodiments, size exclusion filtration is employed to isolatesperm and epithelial cells from a sample (step 3.1). In various sizeexclusion filtration embodiments, the cell suspension can be initiallywashed with washing solution, and the wash can be accomplished directlyon the filter, or in another vessel. The washing solution/cellsuspension mixture, once applied to the size exclusion filter, iscentrifuged. The filtrate, containing impurities that were bound to thecells, is then removed. The cells originally present in the sample areisolated on the filter. In various specific embodiments, the spin filterused is a Corning spinX Costar 8160 filter, with a pore size of 220 nm,available from Sigma (part # CLS8160). Other spin filters with similarpore sizes can also be employed. The pore size of the filter should notbe larger than 2 μm, in order to keep epithelial cells from passing intothe filtrate.

In some embodiments, size exclusion filtration is employed to isolatesperm and epithelial cells from a sample with swab in the wash solution(step 3.1). The washing solution/cell suspension mixture and swab, onceapplied to the size exclusion filter, is centrifuged. The filtrate,containing impurities such as extraneous DNA, is then removed. The cellsoriginally present in the sample are isolated on the filter. The swabcontaining trapped cells is also retained on the filter. In variousspecific embodiments, the spin filter used is a Corning spinX Costar8160 filter, with a pore size of 220 nm, available from Sigma (part #CLS8160). Other spin filters with similar pore sizes can also beemployed. The pore size of the filter should not be larger than 2 μm, inorder to keep epithelial cells from passing into the filtrate.

Although centrifugation, size exclusion filtration (usingcentrifugation) and particle trapping are described here for isolatingcells, the present invention is not limited to these techniques. Forexample, cells can be isolated by the methods described in U.S.Published Application No. 2005/0032097 (“the '097 Publication”),incorporated by reference in its entirety. The '097 Publicationdescribes a process for the isolation of sperm DNA from a samplecontaining both sperm and non-sperm cells. Briefly, selective lysis ofnon-sperm cells is carried out initially, with SDS and proteinase K.Sperm cells are then isolated from the non-sperm cell lysate by vacuumfiltration using a 2 μm pore size. The filtrate is then removed andsperm cells are digested on the filter using a reducing agent such asβ-mercaptoethanol, dithiothreitol, reduced glutathione, or combinationsthereof. The sperm DNA is then isolated from the solubilized sperm.

In other processes, antibody coated particles can be used to bind cellsurface proteins distinct to one type of cell. In addition, flowcytometry and vacuum filtration can be used to selectively isolatecells.

Once cells are isolated from the remainder of the cell suspensionmedium, another wash of the cells can be employed. In variousembodiments, the washing solution is salt solution such as MgCl₂ dilutedin 1×PBS. In these embodiments, the final concentration of MgCl₂ canrange from about 20 mM to 300 mM. This MgCl₂ concentration range canalso be used for the initial wash step 2. In various embodiments, TURBO™DNase, available from Applied Biosystems and Ambion® (Foster City,Calif., part # AM2238), or DNase I supplied by Ambion®(Catalog# AM222)is employed as in washing solution to digest the extraneous DNA.Alternatively, other commercially available DNase enzymes can be used.

In some wash solution embodiments, DNase is used in conjunction withMgCl₂, in the same solution. In other embodiments, a wash with acationic salt (e.g., MgCl₂) (step 2 or 4 in FIG. 1) is immediatelyfollowed by a wash with a DNase enzyme (e.g., by carrying out both steps2 and 4 in FIG. 1, or if step 2 is omitted, by repeating steps 3 and 4twice). In yet other embodiments, a DNase wash (e.g., step 2 or 4 inFIG. 1) is followed by a wash with a cationic salt.

In some embodiments, the washing solution comprises heparin or otherpolyanions such as poly(acrylic acid), poly(methacrylic acid),poly(styrene sulfonate), poly(2-acrylamido-2-methyl-1-propane-sulfonicacid) (PAMPS), or NAFION® (DuPont™), diluted in a physiologicallyacceptable buffer. In some embodiments, the washing solution comprises apolycation such as poly-lysine. Still, in some embodiments, the washsolution can comprise DNA from a non-primate source, to compete thefemale epithelial DNA from a sperm cell's surface. In variousembodiments, the wash is carried out in one or multiple serial stepswith the same or different washing solutions.

In certain embodiments using centrifugation, cells are pelleted prior toa wash step and the cell pellet is washed by resuspending it in washingsolution, (see FIG. 1, step 4) to selectively lyse leukocytes if presentand disrupt any cell-impurity, or specifically cell-nucleic acidinteractions that may exist. The washing solution, in some embodiments,comprises MgCl₂, KCl or NaCl. The salt concentration is high enough toachieve the desired function of breaking leukocytes and to disengage DNAfrom the sperm and epithelial cells' surfaces. Once the wash iscompleted, the cells are recentrifuged to form a washed cell pellet andthe supernatant containing spent washing solution is removed. Removal ofthe supernatant can be done manually or in an automated fashion. Cellwashing, recentrifugation, and wash buffer removal can be repeated, ifdesired. If these steps are repeated, the wash buffer can be the same ordifferent. In the present methods, centrifugation is typically carriedout at 18 k RCF for 1 min., although other conditions are within thescope of the invention.

In certain embodiments using size exclusion filtration, a washingsolution is applied to the filter where the cells are captured. Thewashing solution is pipetted up and down onto the filter to dislodge thecapture cells and to insure all sides of the cells are washed. Once thecells are mixed with washing solution, a centrifugation step is employedto recapture the cells (now washed) on the size exclusion filter. Thefiltrate, containing spent washing solution, is discarded. These stepsencompass step 4 in FIG. 1.

In certain embodiments using magnetic particles, a wash is carried outby mixing a washing solution with the isolated cells and magneticparticles. For mixing to be complete, the magnetic field should beremoved from the vicinity of the vessel wherein the reaction is takingplace. Once mixing is accomplished, the magnetic field can be reappliedto the vessel to sequester the cells and particles from the spentwashing solution. The spent washing solution is then removed.

In some embodiments, and regardless of the method used for cellisolation, a wash with multiple components is employed (step 2 or 4,FIG. 1). For example, a first component can be an MgCl₂ wash orpolyanion wash or polycation wash, or a combination thereof and a secondcomponent can be a DNA degradation step with a DNase enzyme. Thesecomponents can be mixed and then added to the sample.

Any DNase that does not lyse sperm cells partially or in their entirety,can be employed. DNase, present in a DNase reaction buffer (e.g., Turbo™DNase from Applied Biosystems and Ambion®), can be added and mixed withthe sequestered cells as described above for other wash embodiments.DNase in its buffer is added to the sequestered cells, and mixed. In theparticle embodiments, the magnetic field is removed from the vessel uponmixing. The magnetic field is then reapplied, or a centrifugation stepemployed, depending on the particular embodiment, to sequester the cellsfrom the now contaminated (i.e., spent) washing solution. Any remainingDNase, along with the DNase reaction buffer (with degraded DNA present),is then removed in an automated fashion or manually, by pipetting oraspirating. One or more additional washing solutions are then added tothe sequestered cells in a serial manner, if desired.

In other embodiments, a wash step is performed prior to and after DNasetreatment of the sample. In these embodiments, the first wash step canbe accomplished before and/or after initial cell sequestration (i.e., atstep 2 or 4). In these embodiments, a minimum of three serial washes arecarried out.

Once the cells are washed and isolated (regardless of method) from thespent washing solution (i.e., cell suspension medium), the sperm cellsare lysed (FIG. 1, step 5).

Selective sperm lysis buffers that can be used in certain embodimentsare disclosed in co-pending commonly owned U.S. Prov. App. Ser. No.60/899,106, incorporated by reference in its entirety.

In some embodiments, selective sperm cell lysis buffers comprise atleast one disulfide bond reducing reagent (e.g., dithiothreitol (DTT),tris(2-carboxyethyl)phosphine HCl (TCEP), mercaptoethanol (ME),glutathione (GSH)) and at least one salt reagent (e.g., sodium chloride(NaCl), potassium chloride (KCl), lithium chloride (LiCl), magnesiumchloride (MgCl₂), magnesium sulfate (MgSO₄), sodium nitrate (NaNO₃),calcium chloride (CaCl₂), calcium sulfate (CaSO₄)). Other spermselective lysis buffers, e.g., 10% β-mercaptoethanol combined with 2 MDTT, can be employed.

The concentration of the salt reagent in the selective sperm lysisbuffer can be at least 0.1 M. 0.5 M, 1 M, 2 M or higher. Theconcentration of the disulfide bond reducing reagent can be at least0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.7 M or 0.8 M.

In particular embodiments selective sperm lysis buffers for use with thepresent invention include, but are not limited to: (1) 880 mM NaCl and85 mM DTT prepared in 1×PBS, (2) 700 mM KCl and 85 mM DTT prepared in1×PBS and 260 mM Mg₂Cl and (3) 85 mM DTT prepared in 1×PBS.

Based on the components taught for the selective sperm lysis buffer, oneof skill in the art can optimize the final concentration of the salt anddisulfide bond reducing reagent to lyse the sperm cells while keepingthe non-sperm cells essentially intact. One nonlimiting example ofselective sperm lysis buffer is buffer comprising 200 mM DTT and 1 MKCl. Other combinations of a disulfide bond reducing reagent and salt,as described supra may be used at these respective concentrations. Insome embodiments, more than one salt can be employed with one disulfidebond reducing agent. In various embodiments, more than one disulfidebond reducing agent is employed with one salt.

In various embodiments of the invention, mixing the selective spermlysis buffer with the sequestered cells can be accomplished by pipettingup and down into the vessel. In some embodiments, the selective lysisbuffer and cell population are combined and vortexed at a low speed, inorder to allow for full interaction between cells and lysis buffer. Insome embodiments, the cells are lysed by diffusion. Lysis bufferconditions and type of mixing employed influence the duration of thelysis step. These conditions can be optimized using no more thanordinary skill and optimization is dependent on the particularapplication (e.g., forensic or not, types of cells involved etc.) of thepresent methods. In some embodiments, this step, and the other steps ofthe present invention, are carried out in an automated fashion.

In various particle embodiments, selective sperm lysis buffer is addedbefore discontinuation of the magnetic field. In some embodiments,selective lysis buffer is added after discontinuation of the magneticfield.

In certain embodiments, the components of the selective sperm lysisbuffer are added sequentially with the isolated cells and mixedone-by-one with the cells. Alternatively, mixing does not occur untilboth components (i.e., the salt and disulfide bond reducing agent) areadded. In some embodiments, the disulfide bond reducing agent is addedfirst. In some embodiments, lysis occurs by passive diffusion and,therefore, mixing is not performed by the user. In various embodiments,mixing occurs by a combination of passive mixing and active mixing(e.g., by vortexing or pipetting).

In various embodiments, incubation with selective sperm lysis buffer iscarried out at any temperature for a length of time sufficient toachieve the appropriate results. In certain embodiments, the incubationis carried out at room temperature. Alternatively, the incubation can becarried out at approximately 20-50° C. The incubation interval rangesfrom about 1 minute to 4 hours or longer, with more specific typicalintervals being 5 minutes or 10 minutes.

In particle embodiments, the sperm cell lysate can be recovered at step6 from the reaction vessel by reapplying a magnetic field to thereaction vessel, thereby sequestering the cells that were not lysed (andthe particles). The supernatant, after selective lysis, will include thesperm cell lysate, and can be subsequently removed from the vessel. Thiscan be accomplished in an automated fashion, or manually by pipetting.An optional wash can be performed at this stage to further reduce anynucleic acid from the first lysate that would contaminate the intactcells of the epithelial cells, if epithelial cell analysis is desired.

In centrifugation embodiments, selective sperm lysis buffer is added tothe washed cell pellet at step 6, and mixed. Similarly, in sizeexclusion filtration embodiments, selective sperm lysis buffer is addedto the filter and pipetted up and down (either manually or in anautomated fashion), to dislodge the cells from the filter, and to ensureall cells interact with the lysis buffer. Once lysis is complete, acentrifugation step is carried out and the supernatant, containing spermlysate, including sperm nucleic acid (in embodiments usingcentrifugation) or the filtrate, containing sperm lysate, includingsperm nucleic acid (in embodiments using size exclusion) is recovered.Centrifugation, used alone or as part of a size exclusion technique, canbe carried out, e.g., for 1 min. at 9K RCF (i.e., 10,000 RPM in aBeckman Coulter Microfuge 18 centrifuge). For the purposes of thisinvention, any centrifuge amenable to the vessels defined herein can beemployed for the various method steps.

In some embodiments, the DNA, mRNA, or total RNA is purified from thesperm lysate at step 7, in preparation for a downstream application,such as amplification, sequencing or STR analysis, all of which areknown analytical methods. Purification can be employed in any vessel, orpredispensed reagent cartridge. Purification methods are well known inthe art, see, e.g., U.S. Pat. Nos. 5,234,809, and 5,705,628,incorporated in their entirety herein. Extraction with guanidiniumthiocyanate-phenol-chloroform, ethanol, and solid phase binding methodsare all within the scope of the invention. Some solid phases that can beharnessed as substrates for DNA isolation are magnetic, styrene, and/orsilica particles. In addition, nitrocellulose can be used to isolateDNA. Purification can entail mixing the lysate with nucleic acid captureparticles and an appropriate buffer for nucleic acid, or in a variousembodiments, DNA capture.

In various particle embodiments, the particles for cell trapping couldalso be used for DNA purification (step 7) under appropriate conditions.For example, when magnetic silica particles are used to trap cells, thetrapped cells can be lysed and the released DNA can be captured andpurified by the same trapping particles based on standardsilica/chaotropic chemistry (e.g., U.S. Pat. No. 5,234,809 incorporatedby reference in its entirety). In another example, if Dynabeads® MyOnecarboxylic acid beads or magnetic iron oxide particles are used for celltrapping, DNA precipitation chemistry can be used to purify DNA aftercell lysis (see, e.g., U.S. Pat. No. 5,523,231, U.S. Pat. No. 5,705,628both incorporated in their entirety by reference).

In an embodiment where the sperm nucleic acid is purified with magneticparticles, an elution buffer is used to concentrate the nucleic acid insolution, and out of the solid state. Nonlimiting examples of elutionbuffers include deionized water, TE buffer, and any other low-saltbuffer. In addition, heat may be added to the elution reaction duringthis step.

In some embodiments, the sperm lysate can be used for downstreamreactions (e.g., nucleic acid amplification or sequencing), withoutfurther purification.

In certain embodiments, the methods of the present invention furthercomprise isolation and recovery of an epithelial cell's nucleic acid,after removal of the sperm cell's lysate. In some embodiments, theoriginal sample contains sperm and epithelial cells and lysis of theepithelial cells need not be selective. Non-selective lysis may becarried out, for example, with a chaotropic, high salt, ordetergent-based lysis buffer using methods well known in the art. Invarious embodiments, lysis can be carried out by subjecting the cells toheat, in order to break open the cells. Isolation and analysis of theepithelial cells in addition to the sperm cells permits assailant andvictim to be both identified and associated.

In various embodiments of the invention, and in order to lyse a secondspecific cell type, the selective lysis buffer, or a general lysisbuffer (depending on the contents of the original sample and thespecific purpose of nucleic acid recovery) is mixed with the sequesteredcells by pipetting up and down into the vessel. The epithelial cells aresequestered and isolated at step 6, when the sperm cell lysate isrecovered.

In some embodiments, the second lysis buffer and cell population arevortexed, in order to allow for full interaction between cells and lysisbuffer. In some embodiments, the cells are lysed passively by diffusionwithout vortexing or active mixing. Lysis buffer conditions and type ofmixing employed dictate how long the lysis step is carried out for.These conditions can be optimized and are dependent on the particularapplication.

In other embodiments, the epithelial cells are present on a sizeexclusion filter in a vessel, and a lysis buffer is introduced to thetop of the filter. The lysis buffer can then be pipetted up and down todislodge the cells from the filter and to break up the cells, or allowedto incubate on the filter for a period of minutes. The vessel is thencentrifuged and the epithelial cell lysate is present in the filtrate.

In some embodiments, the epithelial lysate is purified in anothervessel, or specifically a compartment of a predispensed reagentcartridge, with magnetic particles and an appropriate buffer (suitablefor binding of nucleic acid to particles). This step is accomplished ina similar fashion to the embodiment describing the isolation of thenucleic acid of the first cell type.

In embodiments using particles, the nucleic acid (either DNA or RNA,preferably DNA) can be isolated further from the epithelial or spermlysate using the same particles originally used to sequester the cellpopulations. DNA is purified by the particles in the original vessel bychanging the buffer conditions depending on the types of magneticparticles used for sequestering (e.g., by adding heat, salt, increasingor decreasing pH, etc.) in the vessel. For example, if the sequesteringparticles are magnetic silica particles, DNA can be captured by theparticles in the original vessel by adding 5M GuSCN (guanidinethiocyanate) solution to the vessel. DNA on the particles is then washedwith ethanol. Elution of the nucleic acid can then be carried out asdescribed above. Alternatively, DNA isolation from the cell lysate canbe carried out by other commercially viable protocols, such as thosesupplied by Promega (Madison, Wis.) under the trade names DNA IQ™.

In some embodiments, the second cell lysate is also removed from thevessel, either in an automated fashion, or manually, as described supra.The lysate can then be introduced into downstream assays that are wellknown in the art, for example, nucleic acid purification, amplification,sequencing or STR typing.

In various embodiments, the components to carry out selective spermlysis, and recovery of its nucleic acid, in a sample containing morethan one cell type, are supplied as a kit. In particle embodiments, aspecific kit includes at least one reaction vessel, a quantity ofparticles responsive to a magnetic field, predetermined to be sufficientto sequester cells in a sample containing a specific quantity of cells,a quantity of washing solution sufficient to wash at least cells of onecell type, a quantity of selective lysis buffer sufficient to lyse thecells of the first type, a quantity of a general or selective lysisbuffer, sufficient to lyse cells of the second cell type, andinstructions for use. In some embodiments, the washing solution is MgCl₂diluted in 1×PBS with a final MgCl₂ concentration of 260 mM. In otherembodiments, the washing solution is comprised of components used in aserial manner, e.g., DNase supplied in a reaction buffer, and/or 1×PBS,1×MgCl₂ or polyanion or polycation. The present method can be practicedwith existing instrumentation which provides a source of a magneticfield. Sequestration of cells can be discerned visually.

In some non-particle embodiments, a specific kit includes at least onereaction vessel, optionally a size exclusion filter that fits in thereaction vessel, a quantity of washing solution sufficient to wash atleast cells of one cell type, a quantity of selective lysis buffersufficient to lyse the sperm cells, a quantity of a general or selectivelysis buffer, sufficient to lyse epithelial cells, and instructions foruse. In some embodiments, the washing solution is MgCl₂ diluted in1×PBS. In other embodiments, the washing solution is comprised ofcomponents used in a serial manner, and can be selected from DNasesupplied in a reaction buffer, 1×PBS, 1×MgCl₂, polyanions andpolycations. However, the present invention is not limited to these washcomponents.

The present invention is further illustrated by reference to theExamples below. However, it should be noted that the Examples, like theembodiments described above, are illustrative and are not to beconstrued as restricting the enabled scope of the invention in any way.

Example 1 Comparing Differential Extraction Procedures: The Prior ArtWashing Solution (1×Pbs) Vs. MgCl₂ Washing Solution

500 μL MgCl₂ washing solution (260 mM final concentration, in 1×PBS) or1×PBS was added to cell suspension samples containing 50 μL of aheterogeneous cell suspension, containing approximately 10,000epithelial cells, 1,000 sperm cells and impurities, including residualDNA. The samples with the washing solutions were then transferred tospin filter tubes (Corning spinX Costar 8160, pore size of 220 mm). Thetubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18centrifuge, after which, the filtrate was discarded.

200 μL selective sperm lysis buffer, comprising 875 mM NaCl₂ and 85 mMdTT, diluted in 1×PBS was added to the spin filter tubes. Sperm cellswere then lysed by mixing, for approximately 5 minutes. The tubes werecentrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18 centrifugeto separate the sperm lysates from intact epithelial cells.

Next, sperm DNA purification was carried out using Applied Biosystems'proprietary DNA purification method. DNA can also be purified usingcommercially available kits such as DNA IQ™ from Promega. After DNApurification, the sperm DNA was quantified using the Quantifiler® YHuman Male DNA Quantification Kit (Applied Biosystems, Foster City,Calif.) and the epithelial DNA is quantified using the Quantifiler®Human DNA Quantification Kit (Applied Biosystems, Foster City, Calif.).

Approximately 1 ng of sperm or epithelial DNA was used for eachamplification reaction. STR amplification was carried out usingAmpFISTR® Indenfiler® PCR Amplification Kit (Applied Biosystems, FosterCity, Calif.) on a GeneAmp® PCT System 9700 (Applied Biosystems, FosterCity, Calif.) according to the manufacturer's protocol.

STR typing was carried out using ABI PRISM 3100 Genetic Analyzer anddata was analyzed using GenMapID3.2.

Results of sperm cell analyses are given in FIGS. 2A-2D, along with STRtyping of control samples of sperm DNA and epithelial DNA. Therespective loci names are listed below the corresponding peaks. Peaksare labeled E or S for specific epithelial cell and sperm cell peaks,respectively. As can be seen in these Figures, a MgCl₂ wash serves toeliminate contamination of epithelial cell DNA, more so than a 1×PBSwash. The STR peaks for the epithelial fraction are higher for the 1×PBSwash than for the MgCl₂ wash.

Example 2 MgCl₂ Washing Solution Reduces Extraneous Epithelial DNA froman Epithelial Cell Sample

It has been found that MgCl₂ in a range of concentrations from zero to260 mM is effective in removing surface bonded DNA more so than when aclassical washing solution, 1×PBS, is used (FIG. 3). The epithelial cellsamples were first washed with MgCl₂, diluted in 1×PBS and then mixed.Wash buffer was removed and the cells were incubated with a selectivesperm lysis buffer. Lysis was implemented for 5 minutes, after which thesample was filtered and the DNA in the filtrate was purified andquantified. Because the lysis buffer is selective, what is left in thefiltrate is residual epithelial DNA originally present in the sample. Ascan be seen in FIG. 3, the amount of residual DNA is reduced with anMgCl₂ concentration as low as 30 mM, as compared to the 1×PBS control.The optimal MgCl₂ concentration, as evident from FIG. 3, is between 160mM and 260 mM.

Example 3 Comparing Differential Extraction Procedures: No DNase WashVs. DNase Wash

Five hundred μL MgCl₂ washing solution (260 mM final concentration, in1×PBS) was added to cell suspension samples containing 50 μL ofpost-coital samples. After mixing and incubation at room temperature for5 minutes, the samples with the washing solutions were transferred tospin filter tubes (Corning spinX Costar 8160, pore size of 220 nm). Thetubes were centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18centrifuge, after which, the filtrate was discarded.

One hundred μL DNase wash solution, containing 10 units of Turbo DNase(AM2238, Applied Biosystems, Foster City, Calif.), was added to one ofthe two samples in the spin filter tubes. DNase wash was carried out at37° C. for 5 minutes. The tubes were centrifuged at 14,000 RPM in aBeckman Coulter Microfuge 18 centrifuge, after which, the filtrate wasdiscarded. The application of the two wash solutions (MgCl₂ and DNase)corresponds to step 2 in FIG. 1.

Next, 200 μL selective sperm lysis buffer, comprising 875 mM NaCl₂ and85 mM dTT, diluted in 1×PBS was added to the spin filter tubes. Spermcells were then lysed by mixing, for approximately 5 minutes. The tubeswere centrifuged at 14,000 RPM in a Beckman Coulter Microfuge 18centrifuge to separate the sperm lysates from intact epithelial cells.

Next, sperm DNA purification was carried out using Applied Biosystems'proprietary DNA purification method. DNA can also be purified usingcommercially available kits such as DNA IQ™ from Promega. After DNApurification, the sperm DNA was quantified using the Quantifiler® YHuman Male DNA Quantification Kit (Applied Biosystems, Foster City,Calif.) and the epithelial DNA is quantified using the Quantifiler®Human DNA Quantification Kit (Applied Biosystems, Foster City, Calif.).

Approximately 1 ng of sperm or epithelial DNA was used for eachamplification reaction. STR amplification was carried out usingAmpFISTR® Indenfiler® PCR Amplification Kit (Applied Biosystems, FosterCity, Calif.) on a GeneAmp® PCT System 9700 (Applied Biosystems, FosterCity, Calif.) according to the manufacturer's protocol.

STR typing was carried out using ABI PRISM 3100 Genetic Analyzer anddata was analyzed using GenMapID3.2.

Results of sperm cell analyses are given in FIGS. 4A-4D. The respectiveloci names are listed below the corresponding peaks. Peaks are labeled Eor S for specific epithelial cell and sperm cell peaks, respectively. Ascan be seen in these Figures, a DNase wash after a MgCl₂ wash serves toeliminate contamination of epithelial cell DNA, more so than an MgCl₂wash without a DNase wash. The STR peaks for the epithelial fraction arehigher for no DNase wash than for the DNase wash.

The invention is further described below by reference to claims asfollows:

I claim:
 1. A method for selectively recovering DNA from sperm cellsfrom a sample containing intact sperm cells and intact epithelial cells,the method comprising the steps of: (a) washing a sample comprisingintact sperm cells and intact epithelial cells by mixing the sample witha washing solution, wherein the washing solution comprises from 30 mM to260 mM MgCl₂ and thereby forming a washed sample; (b) applying DNase tothe washed sample and thereby forming a DNased sample; (c) applying aselective sperm lysis buffer comprising NaCl and DTT to the DNasedsample of step (b); and (d) isolating sperm DNA from the sample of step(c).
 2. The method of claim 1, wherein 0.01 U to 50 U of DNase per 100uL is applied to the washed sample.
 3. The method of claim 1, comprisingpurifying the isolated sperm DNA to a purity sufficient for sperm shorttandem repeat (STR) profiles to be obtained that are suitable forgenotyping.
 4. The method of claim 1, wherein the washing solutioncomprises from 160 mM to 260 mM MgCl₂.